Assay for non-peptide agonists to peptide hormone receptors

ABSTRACT

The invention features a method for determining whether a candidate compound is a non-peptide agonist of a peptide hormone receptor. In this method, a candidate compound is exposed to a form of the peptide hormone receptor which has an enhanced ability to amplify the intrinsic activity of a non-peptide agonist. The second messenger signaling activity of the enhanced receptor is measured in the presence of the candidate compound, and compared to the second messenger signaling activity of the enhanced receptor measured in the absence of the candidate compound. A change in second messenger signaling activity indicates that the candidate compound is an agonist. An increase in second messenger signaling activity indicates that the compound is either a full or partial positive agonist; a decrease in second messenger signaling activity indicates that the compound is an inverse (also termed a ‘negative’) agonist.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of the filing date of U.S. Ser.No. 08/570,157, filed Dec. 11, 1995, now U.S. Pat. No. 5,750,353.

This invention was made in part with Government funding under NationalInstitute of Health grant #DK46767, and the Government therefore hascertain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to peptide hormone receptors.

Peptide hormone receptors are important targets for drug researchbecause a considerable number of diseases and other adverse effectsresult from abnormal receptor activity. High affinity, high specificity,non-peptide antagonists for peptide hormone receptors have beendeveloped. These antagonists are therapeutically useful for decreasingreceptor activation by endogenous hormones. Developing non-peptideagonists proved to be far more difficult.

One peptide hormone of interest, cholecystokinin (CCK), is aneuropeptide with two distinct receptors: CCK-A and CCK-B/gastrin(Vanderhaeghen et al., Nature, 257:604-605, 1975; Dockray, Nature,264:568-570, 1976; Rehfeld, J. Biol. Chem., 253:4022-4030, 1978; Hill etal., Brain Res., 526:276-283, 1990; Hill et al., J. Neurosci.,10:1070-1081, 1990; Woodruff et al., Neuropeptides, (Suppl.) 19:57-64,1991). The peripheral type receptor CCK-A is located in discrete brainnuclei and, in certain species, the spinal cord, and is also involved ingallbladder contraction and pancreatic enzyme secretion. TheCCK-B/gastrin receptor is most abundant in the cerebral cortex,cerebellum, basal ganglia, and amygdala of the brain, as well as inparietal cells of the gastrointestinal tract. CCK-B receptor antagonistshave been postulated to modulate anxiety, panic attacks, analgesia, andsatiety (Ravard et al., Trends Pharmacol. Sci., 11:271-273, 1990; Singhet al., Proc. Natl. Acad. Sci. U.S.A., 88:1130-1133, 1991; Faris et al.,Science, 219:310-312, 1983; Dourish et al., Eur.J.Pharmacol., 176:35-44,1990; Wiertelak et al., Science, 256:830-833, 1992; Dourish et al.,Science, 245:1509-1511, 1989).

SUMMARY OF THE INVENTION

Applicants have developed a systematic screening assay for identifyingnon-peptide agonists specific to peptide hormone receptors. The assay isbased on applicants' recognition that a peptide hormone receptor havingthe capability of amplifying the intrinsic activity of a ligand isuseful as a screening vehicle to identify receptor-specific agonists. Inaddition, a receptor with a signaling activity higher than thecorresponding human wild-type basal level of signaling activity isespecially useful for detecting a reduction in activity induced by aninverse agonist. In both cases, the receptor amplifies the signalgenerated when the ligand interacts with its receptor, relative to thesignal generated when the ligand interacts with a human wild-typereceptor. Thus, forms of a receptor with the ability to amplify receptorsignaling are useful for efficiently screening positive and inversenon-peptide agonists to the corresponding human wild-type form of thereceptor.

Accordingly, the invention features a method for determining whether acandidate compound is a non-peptide agonist of a peptide hormonereceptor. In this method, a candidate compound is exposed to a form ofthe peptide hormone receptor which has a greater, or an enhanced,ability to amplify the intrinsic activity of a non-peptide agonist(hereafter an ‘enhanced receptor’). The second messenger signalingactivity of the enhanced receptor is measured in the presence of thecandidate compound, and compared to the second messenger signalingactivity of the enhanced receptor measured in the absence of thecandidate compound. A change in second messenger signaling activityindicates that the candidate compound is an agonist. For example, anincrease in second messenger signaling activity indicates that thecompound is either a full or partial positive agonist; a decrease insecond messenger signaling activity indicates that the compound is aninverse (also termed a ‘negative’) agonist.

By “intrinsic activity” is meant the ability of a ligand to activate areceptor, i.e., to act as an agonist. By ‘amplify’ is meant that thesignal generated when the ligand interacts with the enhanced receptor iseither higher for a positive agonist, or lower for an inverse agonist,than the signal produced when the same ligand interacts with acorresponding non-enhanced receptor, e.g., a wild-type human receptor. A‘non-enhanced receptor’, for the purposes of this invention, is awild-type human receptor for the peptide hormone of interest. By“corresponding” is meant the same type of peptide hormone receptoralbeit in another form, e.g., a constitutively active mutant receptor.By way of example, the corresponding wild-type form of a constitutivelyactive mutant CCK-B/gastrin receptor would be a wild-type CCK-B/gastrinreceptor; the human CCK-B/gastrin receptor is the corresponding humanform of the rat CCK-B/gastrin receptor.

Examples of enhanced receptors include synthetic mutant receptors, e.g.,constitutively active mutant receptors; other mutant receptors withnormal basal activity which amplify the intrinsic activity of acompound; naturally-occurring mutant receptors, e.g., those which causea disease phenotype by virtue of their enhanced receptor activity, e.g.,a naturally-occurring constitutively active receptor; and eitherconstitutively active or wild-type non-human receptors, e.g., rat,mouse, mastomys, Xenopus, or canine receptors or hybrid variantsthereof, which amplify an agonist signal to a greater extent than doesthe corresponding wild-type human receptor. An enhanced receptor may,but does not always, have a higher basal activity than the basalactivity of a corresponding human wild-type receptor. Methods formeasuring the activity of an enhanced receptor relative to the activityof a corresponding wild-type receptor are described and demonstratedbelow.

Examples of peptide hormone receptors within the scope of the inventioninclude, but are not limited to, receptors specific for the followingpeptide hormones: amylin, angiotensin, bombesin, bradykinin, C5aanaphylatoxin, calcitonin, calcitonin-gene related peptide (CGRP),chemokines, cholecystokinin (CCK), endothelin, follicle stimulatinghormone (FSH), formyl-methionyl peptides, galanin, gastrin, gastrinreleasing peptide, glucagon, glucagon-like peptide 1, glycoproteinhormones, gonadotrophin-releasing hormone, leptin, luteinizing hormone(LH), melanocortins, neuropeptide Y, neurotensin, opioid, oxytocin,parathyroid hormone, secretin, somatostatin, tachykinins, thrombin,thyrotrophin, thyrotrophin releasing hormone, vasoactive intestinalpolypeptide (VIP), and vasopressin. An enhanced receptor can furtherembrace a single transmembrane domain peptide hormone receptor, e.g., aninsulin receptor.

An “agonist”, as used herein, includes a positive agonist, e.g., a fullor a partial positive agonist, or a negative agonist, i.e., an inverseagonist. An agonist is a chemical substance that combines with areceptor so as to initiate an activity of the receptor; for peptidehormone receptors, the agonist preferably alters a second messengersignaling activity. A positive agonist is a compound that enhances orincreases the activity or second messenger signaling of a receptor. A“full agonist” refers to an agonist capable of activating the receptorto the maximum level of activity, e.g., a level of activity which isinduced by a natural, i.e., an endogenous, peptide hormone. A “partialagonist” refers to a positive agonist with reduced intrinsic activityrelative to a full agonist. As used herein, a “peptoid” is apeptide-derived partial agonist. An “inverse agonist”, as used herein,has a negative intrinsic activity, and reduces the receptor's signalingactivity relative to the signaling activity measured in the absence ofthe inverse agonist. A diagram explaining the difference between fulland partial agonists, inverse agonists, and antagonists is shown in FIG.1 (see also Milligan et al., TIPS, 16:10-13, 1995).

Examples of peptide hormone receptor specific peptide agonists andnon-peptide antagonists useful in the screening assay of the inventionare described below. Non-peptide ligands include, but are not limitedto, the benzodiazepines, e.g., azabicyclo[3.2.2]nonane benzodiazepine(L-740,093; Castro Pineiro et al., WO 94/03437). L-740,093 S andL-740,093 R refer to the S-enantiomer and the R-enantiomer of L-740,093,respectively. Where the peptide hormone receptor is a CCK-A orCCK-B/gastrin receptor, useful peptide agonists include, but are notlimited to, gastrin (e.g., sulphated (“gastrin II”) or unsulphated(“gastrin I”) forms of gastrin-17, or sulphated or unsulphated forms ofgastrin-34), or cholecystokinin (CCK) (e.g., sulfated CCK-8 (CCK-8 s),unsulphated CCK-8 (CCK-8d), CCK-4, or pentagastrin (CCK-5)). Fullagonists of the CCK-B/gastrin receptor include, but are not limited to,CCK-8s, and more preferably gastrin (gastrin I).

In contrast, an “antagonist”, as used herein, refers to a chemicalsubstance that inhibits the ability of an agonist to increase ordecrease receptor activity. A ‘full’, or ‘perfect’ antagonist has nointrinsic activity, and no effect on the receptor's basal activity (FIG.1). Peptide-derived antagonists are, for the purposes herein, consideredto be non-peptide ligands.

The invention also features a method of isolating a form of a peptidehormone receptor suitable for detecting agonist activity of anon-peptide ligand. The method involves (a) exchanging a region of afunctional domain of a first peptide hormone receptor with acorresponding region of a functional domain of a second peptide hormonereceptor, the functional domain being selected from the group consistingof an intracellular loop and adjacent parts of a transmembrane domain;and (b) measuring the ability of the first peptide hormone receptor toamplify an agonist signal relative to a corresponding wild-type humanreceptor, a greater amplification in the first peptide hormone receptorwould indicate that the first peptide hormone receptor is suitable fordetecting agonist activity in a non-peptide ligand. The correspondingregion can be between one and ten amino acids, e.g., a block of five toten amino acids, or up to thirty or a hundred amino acids in length. Thefirst and second peptide hormone receptors are preferably linked todifferent second messenger pathways. Those skilled in the art know whichparticular amino acids of the peptide hormone receptors are consideredto be within extracellular, intracellular (cytoplasmic), ortransmembrane regions of the receptor. For example, extracellular,intracellular, and transmembrane regions of the CCK-B/gastrin receptorare determined by sequence alignment with other receptors (FIG. 2), orby hydropathy analysis (Baldwin, EMBO J., 12:1693-1703, 1993).Conformation receptor modelling is described further below.

Another method of isolating a form of a peptide hormone receptorsuitable for detecting agonist activity in a non-peptide ligand involves(a) constructing a series of mutant forms of the receptor by replacingan original amino acid with another amino acid, i.e., a replacementamino acid; and (b) measuring the ability of the first peptide hormonereceptor to amplify an agonist signal relative to the correspondingwild-type human receptor. An amplification in the first peptide hormonereceptor would indicate that the first peptide hormone receptor issuitable for detecting agonist activity in a non-peptide ligand. Thereplaced amino acid can lie in an intracellular domain of the receptoror in a region of a transmembrane domain flanking an intracellularportion of the receptor, e.g., the intracellular domain-proximal half ofthe transmembrane domain, or within, e.g., 8 or 10 amino acids of theintracellular domain. The replacement amino acid can be of the same typein each of the mutant constructs, or various types of amino acids can besubstituted at random. The replacement amino acid can be of the same ora different charge from the original amino acid, e.g., a negative aminoacid can be exchanged for a positive amino acid, a positive amino acidcan be exchanged for a negative amino acid, or a positive or negativeamino acid can be exchanged for a neutral amino acid. Preferably, thereplacement amino acid is glutamine, glutamic acid, aspartic acid, orserine.

Other terms used in the various embodiments of the invention will beunderstood from the following definitions. For example, by a “peptidehormone” is meant a polypeptide that interacts with a target cell bycontacting an extracellular receptor, i.e., a “peptide hormonereceptor”. A “peptide” is used loosely herein to refer to a moleculecomprised of amino acid residues that are connected to each other bypeptide bonds. A “mutant receptor” is understood to be a form of thereceptor in which one or more amino acid residues in the predominantreceptor occurring in nature, e.g., in a naturally-occurring wild-typereceptor, have been either deleted or replaced with a different type ofamino acid residue. By a “constitutively active receptor” is meant areceptor with a higher basal activity level than the correspondingwild-type receptor, where activity means the spontaneous ability of areceptor to signal in the absence of further activation by a positiveagonist. The basal activity of a constitutively active receptor can alsobe decreased by an inverse agonist. A “naturally-occurring” receptorrefers to a form or sequence of the receptor as it exists in an animal,or to a form of the receptor that is synonymous with the sequence knownto those skilled in the art as the “wild-type” sequence. Those skilledin the art will understand a “wild-type” receptor to refer to theconventionally accepted “wild-type” amino acid consensus sequence of thereceptor, or to a “naturally-occurring” receptor with normalphysiological patterns of ligand binding and signaling. A “secondmessenger signaling activity” refers to production of an intracellularstimulus (including, but not limited to, cAMP, cGMP, ppGpp, inositolphosphate, or calcium ion) in response to activation of the receptor, orto activation of a protein in response to receptor activation, includingbut not limited to a kinase, a phosphatase, or to activation orinhibition of a membrane channel.

“Sequence identity,” as used herein, refers to the subunit sequencesimilarity between two nucleic acid or polypeptide molecules. When agiven position in both of the two molecules is occupied by the samenucleotide or amino acid residue, e.g., if a given position (asdetermined by conventionally known methods of sequence alignment) ineach of two polypeptides is occupied by serine, then they are identicalat that position. The identity between two sequences is a directfunction of the number of matching or identical positions, e.g., if 90%of the positions in two polypeptide sequences are identical, e.g., 9 of10, are matched, the two sequences share 90% sequence identity. Methodsof sequence analysis and alignment for the purpose of comparing thesequence identity of two comparison sequences are well known by thoseskilled in the art. “Biological activity”, as used herein, refers to theability of a peptide hormone receptor to bind to a ligand, e.g., anagonist or an antagonist and to induce signaling.

The invention provides an efficient and rapid assay for identifyingnon-peptide agonists that interact with a peptide hormone receptor. Thenewly identified agonists can serve as therapeutics, or as leadcompounds for further pharmaceutical research. Systematic chemicalmodifications can be made; their effects can be functionally assessed inenhanced receptors according to the method of the invention. Byfollowing such a development strategy the intrinsic activity of newagonists is optimized so as to provide useful therapeutics againstdiseases involving a peptide-hormone receptor.

Also embraced are the various mutant peptide hormone receptors disclosedherein, and their respective nucleic acid coding sequences. Plasmidmanipulation, storage, and cell transformation are performed by methodsknown to those of ordinary skill in the art. See, e.g., Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,NY. 1988, 1995.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

DETAILED DESCRIPTION

We first briefly describe the drawings.

DRAWINGS

FIG. 1 is a schematic diagram showing the relationship between a full orpartial agonist, an inverse agonist, and an antagonist.

FIG. 2 is an illustration showing a multiple alignment of cloned CCKreceptor deduced amino acid sequences mastomys CCK-B (SEQ ID NO: 1), ratCCK-B (SEQ ID NO: 2), human CCK-B (SEQ ID NO: 3), canine CCK-B (SEQ IDNO: 4), human CCK-A (SEQ ID NO: 5), rat CCK-A (SEQ ID NO: 6), andXenopus CCK-XL (SEQ ID NO: 7). ‘A’ marks the position in the hCCK-Areceptor where an E to Q substitution results in an increase in PD135,158 intrinsic activity without increasing basal receptor activity.‘B’ marks the position in the hCCK-B receptor where an L to either S orE substitution results in an increase in basal activity. Thecorresponding L to S in the hCCK-A receptor does not result in anincrease in basal activity. ‘C’ marks the position in the hCCK-Breceptor where a V to E substitution results in an increase in basalactivity. The corresponding I to E substitution in the human CCK-Areceptor does not result in an increase in basal activity. The numberingshown is generic; each receptor is different based on deletions orinsertions.

FIG. 3 is a bar graph showing that the intrinsic activity of peptide,peptide-derived and non-peptide ligands at the wild-type CCK-B/gastrinreceptor (top panel) is amplified in a constitutively active receptormutant (bottom panel).

FIG. 4 is an illustration of inositol phosphate production by thenon-peptide agonist L-740,093. Top panel: L-740,093 S stimulatedinositol phosphate production in COS-7 cells expressing a constitutivelyactive human CCK-B/gastrin receptor. Bottom panel: YM022 antagonizes thepartial agonist activity induced by 10 nM L-740,093-S.

FIG. 5 is an illustration of the inositol phosphate production of thenon-peptide agonist L-740,093-R. Top panel: L-740,093 R inhibits basalinositol phosphate production in COS-7 cells expressing a constitutivelyactive human CCK-B/gastrin receptor. Bottom panel: The inverse agonistactivity induced by 10 nM L-740,093 R is partially abolished by YM022 ina concentration-dependent fashion.

FIG. 6 is a comparison of intrinsic activities of CCK-B/gastrin receptorligands utilizing the wild-type and the constitutively active receptors.Values for all compounds follow a logarithmic-linear correlation(r2=0.93).

FIG. 7 is a competition binding curve showing the extent of ¹²⁵I-CCK-8receptor binding. Binding of ¹²⁵I-CCK-8 to COS-7 cells, transientlytransfected with hCCK-B-pcDNAI is shown in the presence of increasingconcentrations of CCK-8, gastrin I, and CCK-4 (part A) and L-364,718 andL-365,260 (part B).

FIG. 8 is a graph showing second messenger signaling (i.e., mobilizationof intracellular calcium) in COS-7 cells that express the recombinanthuman brain CCK-B receptor with (part A, left panel) and without (partA, right panel) the addition of the calcium chelator, EGTA. This isparalleled by an increased production of inositol phosphate (part B).

FIG. 9 is a schematic representation of the seven transmembrane (TM)domain structure of the human CCK-B/gastrin receptor. The C-terminaldomain of the third intracellular loop is highlighted in black.

FIG. 10 is a bar graph of basal inositol phosphate accumulation in COS-7cells transfected with wild-type CCK-B/gastrin receptor (WT), or withone of two constitutively active mutants (Mut.1, Mut.2).

FIG. 11 is a bar graph showing a functional comparison of the CCKreceptors human CCK-A (hCCK-A), human CCK-B (hCCK-B), dog CCK-B(dCCK-B), mouse CCK-B (mCCK-B), and the mastomys CCK receptor.

Recent drug development efforts have led to the discovery of many smallmolecules which competitively block G-protein coupled peptide hormonereceptors. In contrast, very few non-peptide ligands have beenidentified which activate this family of receptors. Here, Applicantsdemonstrate that chemical modifications of known non-peptide ligands forthe CCK-B/gastrin receptor can interconvert small molecules fromantagonists to either positive agonists or to inverse agonists. Changesin the intrinsic activity of the ligand resulting from suchmodifications were detectable because Applicants designed a screeningassay which employed a constitutively active mutant of the humanCCK-B/gastrin receptor (³²⁵L→E). Several peptide, ‘peptoid’ andbenzodiazepine-based non-peptide ligands were tested in this assay, andevaluated for their abilities to activate the recombinant wild-type orconstitutively active mutant receptor, respectively. Whereas fullagonists had similar signaling efficacy in both receptors when comparedto the intrinsic activity of the peptide agonist CCK-8s, the effect ofligands with lesser intrinsic activity was logarithmically amplified bythe constitutively active mutant receptor. The prototypebenzodiazepine-derived non-peptide ‘antagonist’ L-365,260 barelyincreased basal activity of the wild-type CCK-B/gastrin receptor, butwas identified as a partial agonist using the ³²⁵L→E mutant. Minorchemical modification of L-365,260 resulted in compounds which were pureantagonists (YM022), partial agonists (L-740,093 S) or inverse agonists(L-740,093 R). The drug discovery process for novel non-peptideagonists, including those with reverse intrinsic activity, should beguided by using enhanced receptors, e.g., constitutively active mutantreceptors, in the screening assay so as to expedite identification ofpotential lead compounds.

I. Working Example:

The following example demonstrates the usefulness of an enhanced peptidehormone receptor to screen for non-peptide agonists.

Using a constitutively active mutant of the human CCK-B/gastrin receptorit was discovered that several benzodiazepine-based putative non-peptide‘antagonists’ had detectable intrinsic activity when binding to thisreceptor.

The constitutively active CCK-B/gastrin receptor mutant ³²⁵L→E wastransiently overexpressed in COS-7 cells. The fact that it wasconstitutively active was evident from ligand-independent production ofinositol phosphate; the wild-type receptor, in contrast, exhibits onlyligand-dependent inositol phosphate production. Both mutant and thewild-type receptors induced similar inositol phosphate production whenmaximally stimulated with the peptide agonists CCK-8s or gastrin I (FIG.3). In contrast, only the mutant CCK-B/gastrin receptor alloweddetection of the different degrees of intrinsic activities of threebenzodiazepine-derived compounds, L-740,093 R, YM022 and L-365,260. Eachof these compounds were previously considered prototype non-peptideantagonists of the wild-type CCK-B/gastrin receptor (Castro Pineiro etal., WO 94/03437; Lotti et al., Eur. J. Pharmacol., 162:273-280, 1989;Nashida et al., J. Pharmacol. Exp. Ther., 270:1256-61, 1994; Nashida etal., J. Pharmacol. Exp. Ther., 269:725-31, 1994).

The non-peptide compound L-365,260 had 62% efficacy when compared to thefull agonist CCK-8s, and was on that basis identified as a partialagonist in the ³²⁵L→E constitutively active mutant receptor (FIG. 3,right section). In fact, close re-examination of this compound'sfunction in the wild-type CCK-B/gastrin receptor also revealed a barelydetectable, yet significant, increase in inositol phosphate productionthat had not been seen with the other non-peptide compounds.

From the above results it was concluded that minor changes in thechemical groups attached to the benzodiazepine backbone can result inmarked alterations in intrinsic activity of small non-peptide compounds.The stereochemistry of benzodiazepine-derived CCK receptor ligands isanother feature which can alter binding affinity as well as receptorselectivity (Showell et al. J. Med. Chem. 37:719-721, 1994).

The following additional observations confirmed that differences inligand stereochemistry determine the functional properties of theCCK-B/gastrin receptor specific compounds. For example, it was notedthat L-740,093 S was almost a full agonist in the ³²⁵ L→E CCK-B/gastrinreceptor mutant (FIG. 3). When tested with the human wild-typeCCK-B/gastrin receptor, L-740,093 S functions as a partial agonist (25%efficacy compared with CCK-8s). As such, L-740,093 S is the first knownnon-peptide agonist for the CCK-B/gastrin receptor. The mirror image ofL-740,093 S, L-740,093 R, has properties opposite to those of the Senantiomer. L-740,093 R reduces the basal activity of the constitutivelyactive receptor almost to wild-type levels.

To confirm the functional classification of CCK-B/gastrin receptornon-peptide ligands, basic pharmacologic principles were tested todetermine whether they applied to interactions between the CCK-B/gastrinreceptor and the benzodiazepine-derived agonists and antagonists. Of thecompounds tested, YM022 came closest to being a ‘perfect’ antagonist,with almost no intrinsic activity on either the wild-type or theconstitutively active CCK-B/gastrin receptor. In both the wild-type andthe constitutively active receptors, YM022 blocked CCK-8s inducedinositol phosphate production with almost identical affinity, reflectedby similar pA2 values (9.78 and 9.37, respectively). Consistent with thefunctional classification of L-740,093 S as a non-peptide agonist, theinositol phosphate production induced by this compound could be blockedby YM022 (pA2=9.54; FIG. 4). YM022 was also able to attenuate theinverse agonist activity of L-740,093 R on the constitutively activeCCK-B/gastrin receptor (FIG. 5). In a concentration-dependent manner,YM022 partially restored basal activity to the constitutively activereceptor which had been inhibited by 20 nM L-740,093 R. The fact thatbasal activity was not restored completely is explained by the fact thatYM022 itself is a weak inverse agonist in this mutant rather than a purereceptor antagonist.

The pA2 value measures the functional affinity of a competitiveantagonist. In contrast to IC₅₀ values (50% inhibitory concentration),pA₂ values are independent of which agonist concentrations are used tomeasure antagonist affinities. Ideally, pA₂ values should also beindependent of what specific agonist compounds are tested to assessantagonist affinities. The pA2 value is defined as the negativelogarithm of the specific antagonist concentration which shifts theagonist concentration-response curve by a factor of two to the right. Inother words, in the presence of a given antagonist concentration, onewould need twice as much agonist as would be required in the absence ofantagonist to induce the same effect. pA₂ values of competitiveantagonists are typically assessed by Schift plots, but can also bemeasured by simplified ‘null’ methods (Lazareno et al., Trends inPharmacol. Sci., 14:237-239, 1993).

In addition to non-peptide ligands, the constitutively active mutantreceptor amplified the intrinsic activity of peptide-derived partialagonists (‘peptoids’; Horwell et al., Eur. J. Med. Chem., 30Suppl.:537S-550S, 1995; Horwell et al., J. Med. Chem., 34:404-14, 1991).The peptoids used in the following experiments were derived fromsequential modification of CCK-4. Two prototype ‘peptoid’ compounds, PD135,158 and PD 136,450, were converted from partial agonists in thewild-type to almost full agonists in the constitutively activeCCK-B/gastrin receptor. Thus, peptide-derived as well as non-peptidecompounds have increased efficacy on the constitutively active versusthe wild-type CCK-B/gastrin receptor. Despite these marked alterationsin efficacy, the ratio of wild-type versus mutant receptor affinities,as determined by ¹²⁵I-CCK-8 competition binding experiments, fell withina two-fold range (Table 1 A). There was no apparent correlation betweenthe intrinsic activity of CCK-B/gastrin receptor ligands and potencyshifts between the wild-type and the constitutively active receptors(Table 1 B).

Precedent with the constitutively active CCK-B/gastrin receptorillustrates a new strategy using mutant receptors as a ‘magnifyingglass’ to screen for non-peptide leads with some degree of intrinsicactivity. It should be noted that the constitutively active ³²⁵L→Emutant reliably predicted the intrinsic activity that a compound wouldpossess when stimulating the wild-type receptor (FIG. 6). This was trueover the spectrum of peptide, ‘peptoid’, and non-peptide ligands tested.

The intrinsic activity (percent maximal stimulation of inositolphosphate formation) of all compounds was tested at concentrations thatwere at least 100-fold higher than the corresponding receptoraffinities.

The intrinsic activity of L-740,093 S was comparable to that observedfor the ‘peptoid’ ligand PD 135,158, a compound that has been recentlydemonstrated to be a partial agonist in vivo (Ding et al.,Gastroenterology, 109:1181-87, 1995).

TABLE 1 A) ¹²⁵I CCK-8 binding affinities of tested ligands Wild-typereceptor ³²⁵L→E Mutant Ratio Compound Ki(nM) Ki(nM) (Wild-type/Mutant)Gastrin I 1.35 ± 0.28 0.80 ± 0.16 1.69 CCK-8s 0.12 ± 0.01 0.07 ± 0.011.71 PD 135,158 2.25 ± 0.61 1.01 ± 0.19 2.23 PD 136,450 0.99 ± 0.1  0.59± 0.12 1.68 L-740,093 R 0.19 ± 0.02 0.18 ± 0.04 1.06 YM022 0.07 ± 0.010.08 ± 0.01 0.88 L-364,718 150 ± 42  170 ± 34  0.88 L-365,158 7.16 ±0.87 7.83 ± 1.51 0.91 L-740,093 S 19.5 ± 1.5   16 ± 1.4 1.22 B)Signaling potencies of tested ligands Wild-type ³²⁵L→E receptor MutantIC50(nM) IC50(nM) Ratio Compound 95% C.I. 95% C.I. (Wild-type/Mutant)Gastrin I 0.24 (0.12-0.48) 0.20 (0.06-0.69) 1.20 CCK-8s 0.14 (0.11-0.19)0.15 (0.08-0.30) 0.93 PD 135,158 1.05 (0.29-3.83) 1.01 (0.21-4.80) 1.04PD 136,450 0.36 (0.04-3.35) 0.58 (0.23-1.46) 0.62

II. Receptor Binding and Activity Assays:

A. Receptor Binding Assays:

The binding of a ligand to a CCK receptor, e.g., the CCK-A or theCCK-B/gastrin receptor, can be measured according to the followingexample. In this example, the binding affinity of a ligand to the humanCCK-B/gastrin receptor is measured.

COS-7 cells (1.5×10⁶) were plated in 10-cm culture dishes (Nunc) andgrown in Dulbecco's modified Eagle's medium containing 10% fetal calfserum in a 5% CO₂, 95% air incubator at 37° C. After an overnightincubation, cells were transfected (Pacholczyk et al., Nature350:350-354, 1991) with 5-7 μg of a pcDNA I expression vector containinghCCKB (HCCKB-pcDNA I). Twenty-four hours after transfection cells weresplit into 24-well dishes (2×10⁴ cells/well) (Costar). After anadditional 24 hours, competition binding experiments were performed inHank's buffer supplemented with 25 mM phenylmethylsulfonyl fluoride(PMSF). Twenty pM of ¹²⁵I CCK-8 (DuPont-New England Nuclear) was used asradioligand. Equilibrium binding occurred after incubation for 80 min.at 37° C. Cell monolayers were then washed three times, hydrolyzed in 1N NaOH, and the amount of radioactivity to the receptor was quantified.Unlabeled agonists (e.g., CCK-8s, unsulphated CCK-8 (CCK-8 us), gastrinI, CCK-4 (Peninsula)) and antagonists (L364,718 and L365,260 (Merck))were tested over the concentration range of 0.1 pM to 10 μM. All bindingexperiments were repeated three to five times.

The competition data were analyzed using computer software which isspecifically designed for the purpose of radioligand binding assays(Inplot 4.0, GraphPad, San Diego, Calif.). Analyses of competition andsaturation binding data can also be performed using computerizednon-linear curve fitting (McPherson, G. A., J Pharmacol Methods,14:213-28, 1985).

The affinities of all agonists and antagonists were confirmed byrepeating the above assay using Chinese hamster ovary (CHO) cells stablytransfected with human CCK-B/gastrin receptor cDNA. This CHO cell linewas established by transfecting a hCCKB-pcDNAI Neo expression vector(Invitrogen) into CHO cells using a standard lipofection protocol(Bethesda Research Laboratories) followed by G418 selection.

Where binding parameters are determined in isolated plasma membranes,binding can be performed, e.g., for 60 min. at 22° C. (Kopin et al.,Proc. Natl. Acad. Sci. USA, 89:3605-09, 1992). Separation of bound andfree radioligand can be achieved by receptor-binding filtermatfiltration (Klueppelberg, U. G., et al., 1989, Biochemistry 28:3463-8).

In order to compare the binding specificity of CCKB/gastrin mutantreceptors of the invention with the binding specificity typical ofwild-type CCK-B/gastrin receptors see Matsumoto et al. (Am J Physiol.,252:G143-G147, 1987) and Lee et al. (J. Biol. Chem., 268(11):8164-69,1993).

Comparison of binding affinity to that of a wild-type human CCK-Breceptor: A base line value for binding of a radiolabelled ligand to ahuman wild-type receptor, e.g., the human CCK-B/gastrin receptor wasdetermined (see Lee et al., J. Biol. Chem., 268(11):8164-69, 1993).Agonist affinities of the human brain CCK-B/gastrin receptor expressedin COS-7 cells were characterized (FIG. 7). The structurally relatedagonists CCK-8s, gastrin I, and CCK-4 all competed in aconcentration-dependent manner for binding of ¹²⁵I-CCK-8 to COS-7 cellsexpressing the recombinant receptor. The calculated IC₅₀ values forCCK-8s, gastrin I, and CCK-4 are 0.14, 0.94, and 32 nM respectively(FIG. 7, part A). Similar ¹²⁵I-CCK-8 competition curves were assessedwith L-364,718 and L365,260 (FIG. 7, part B), and revealed IC₅₀ valuesof 145 and 3.8 nM, respectively. Untransfected cells showed nodisplaceable binding.

B. Receptor Signaling Activity Assays:

Binding of an agonist to a CCK receptor elicits an increase in theintracellular calcium concentration and in phosphatidylinositolhydrolysis.

Measurement of [Ca²⁺]: Forty-eight hours after transfection withhCCKB-pcDNAI, COS-7 cells were loaded with the Ca²⁺ fluorophore fura-2in modified Krebs-Ringer bicarbonate buffer. Changes in the fluorescenceemission ratios (340:380 nm) after stimulation of cells with 10⁻⁷ MCCK-8s or 10⁻⁶ M gastrin I were measured as previously described (Rajanet al., Diabetes, 38:874-80, 1989). Extracellular calcium can bechelated with EGTA (2.5 mM) to confirm that a gastrin-induced increasein [Ca²⁺] originates primarily from intracellular [Ca²⁺] pools.

Measurement of Inositol phosphate Metabolites: COS-7 cells transfectedwith hCCKB-pcDNAI were cultured in inositol-free Dulbecco's modifiedEagle's medium (DMEM, GIBCO) which was supplemented with 10 μCi/ml[³H]myo-inositol (ARC) for 24 hours prior to analysis. After 1 hour ofequilibration in modified Krebs-Ringer bicarbonate, the cells werestimulated with 10⁻⁷M CCK-8s for 10 seconds and harvested inmethanol-HCl. The aqueous phase was extracted with chloroform,lyophilized, and analyzed for inositol 1,4,5-triphosphate(Ins-1,3,4,5-P₃) and inositol 1,3,4,5-tetrakisphosphate (Ins-1,3,4,5-P₄)by strong anion-exchange high performance liquid chromatography (Augeret al., Cell, 57:167-75, 1989).

Comparison of signaling activity to that of a wild-type human CCK-Breceptor: A baseline level of human wild-type CCK-B receptor secondmessenger signaling activity was measured in response to CCK-8sstimulation of COS-7 cells expressing the receptor (FIG. 8; see Lee etal., J. Biol. Chem., 268(11):8164-69, 1993). CCK-8s (10⁻⁷M) triggered amarked increase in free cytosolic calcium, [ca²⁺]_(i) (FIG. 8, part A,left panel). There was no change in free cytosolic calcium in cellstransfected with the empty expression vector, pcDNAI. After chelation ofextracellular calcium (1.5 mM Ca²⁺ in the buffer) by 2.5 mM EGTA,addition of CCK-8s (10⁻⁷ M) still transiently increased [Ca²⁺]_(i) (FIG.8, part A right panel), suggesting that the initial peak of theCCK-induced increase in [Ca²⁺]_(i) originated primarily fromintracellular Ca²⁺pools. The arrows indicate the addition of CCK-8s (0.1μM) or EGTA (2.5 mM). The pattern of [Ca²⁺]_(i) response suggests thatthe binding of CCK-8s to the recombinant receptor triggers intracellularsignaling through activation of phospholipase C. This was confirmed bymeasurement of inositol phosphate metabolites in hCCKB-pcDNAI-transfected COS-7 cells 10 seconds after CCK-8s stimulation (FIG. 8,part B). This time point was chosen because it immediately precedes theCCK-8-induced [Ca²⁺]_(i) peak. CCK-8s (10⁻⁷M) increased the level ofIns-1,4,5-P₃ by 453% over control, unstimulated hCCKB-pcDNAI-transfected COS-7 cells (n=3, p<0.001). The level of Ins-1,3,4,5-P₄,an immediate metabolite of Ins-1,4,5,-P3, also increased by 186% overcontrol (n=3, p<0.01).

A simplified method for measuring total inositol phosphate content:While the above method specifically assesses Ins(1,4,5)P₃ content, asimplified screening method can be used to test for the totalconcentration of inositol phosphate; the simplified method does notdistinguish between specific isoforms. (This method was used to measureinositol phosphate generation for the experiments shown in FIGS. 3, 4,5, 6, and 10.)

COS-7 cells transfected with receptor cDNA-pcDNAI were cultured ininositol-free, serum-free Dulbecco's modified Eagle's medium (DMEM,GIBCO), supplemented with 3 μCi/ml ³ H-myo-inositol (NEN, 45-80Ci/mmol), for 18 hours prior to analysis. The cells were then washedtwice with DMEM/10 mM LiCl₂ and twice with phosphate-buffered saline/10mM LiCl₂. After stimulation with putative agonists in phosphate-bufferedsaline 10/mM LiCl₂ for 30 minutes at 37° C., cells were scraped inice-cold methanol. Lipids were extracted with chloroform (Pfeiffer etal., FEBS Lett., 204:352-356, 1986). The upper phase was analyzed forinositol phosphates by strong anion exchange chromatography, using Dowex1-X8 columns (BIORAD) and differential elution with water/60 mM ammoniumfornate/2 M ammonium fornate. Eluted radioactivity was measured byliquid scintillation counting, and inositol phosphate content wasexpressed as a percentage of total ³H-radioactivity applied to thecolumns.

Further information on the second messenger pathways linked to thenative parietal cell gastrin receptor can be obtained in the followingreferences: Muallem, S. et al., 1984, Biochim Biophys Acta 805:181-5;Chew, C. S. et al., 1986, Biochim Biophys Acta 888:116-25; Roche, S. etal., 1991, FEBS Letts., 282:147-51.

In addition to inositol phosphate production, second messenger signalingactivity can be measured according to, e.g., cAMP, cGMP, ppGpp, orcalcium ion production, or using as indicators, e.g., intracellular pH,pH-sensitive dyes, or expression of a reporter gene, e.g., a luciferasegene, or measuring channel activity or cell depolarization orhyperpolarization by electrophysiological techniques.

III. Suitable Peptide Hormone Receptors with the Ability to Amplify theIntrinsic Activity of a Non-peptide Agonist:

The screening assay of the invention can be performed using peptidehormone receptors that have a higher activity than the correspondinghuman wild-type receptor. An enhanced basal activity amplifies theintrinsic activity of ligands, and is useful for detecting eitheractivation of the receptor by a partial agonist, or inhibition by aninverse agonist. Receptors that do not have an enhanced basal activityrelative to the corresponding wild-type receptor, but still amplify theintrinsic activity of a partial agonist, are also useful.

Examples of peptide hormone receptors that are useful for screeningnon-peptide agonists include various forms of the receptors thatinteract with the following peptide hormones (along with references fortheir respective wild-type amino acid sequences): amylin, angiotensin,bombesin, bradykinin, C5a anaphylatoxin, calcitonin, calcitonin-generelated peptide (CGRP), chemokines, cholecystokinin (CCK), endothelin,follicle stimulating hormone (FSH), formyl-methionyl peptides, galanin,gastrin, gastrin releasing peptide, glucagon, glucagon-like peptide 1,glycoprotein hormones, gonadotrophin-releasing hormone, leptin,luteinizing hormone (LH), melanocortins, neuropeptide Y, neurotensin,opioid, oxytocin, parathyroid hormone, secretin, somatostatin,tachykinins, thrombin, thyrotrophin, thyrotrophin releasing hormone,vasoactive intestinal polypeptide (VIP), and vasopressin. An enhancedreceptor can further embrace a single transmembrane domain peptidehormone receptor, e.g., an insulin receptor. The wild-type amino acidsequences of the above peptide hormone receptors is available in, and/orreferenced in, Watson and Arkinstall, The G-Protein Linked Receptor,Academic Press, NY., 1994.

Forms of a peptide hormone receptor that are capable of amplifying theintrinsic activity of an agonist include, but are not limited to, thefollowing forms of receptors:

1. Mutant peptide hormone receptors that are capable of amplifying theintrinsic activity of partial agonists.

An example is given of a mutant human CCK-A receptor that enhances theintrinsic activity of the partial ‘peptoid’ agonist PD 135,158, yetcauses no apparent increase in agonist-independent basal receptoractivity, is the mutant CCK-A receptor pMHA35. pMHA35 was made byreplacing amino acids 138-ERY-140 of the human wild-type CCK-A receptorwith QRY in the vector pcDNAI. (See FIG. 2 for an illustration of thewild-type CCK-A receptor amino acid sequence.)

2. CCK-A receptors in which one or more of residues ¹³⁸E, ³⁰⁵L, and ³¹²Iare replaced with any other amino acid residue, e.g., a serine, asparticacid, glutamine, or glutamic acid residue.

3. CCK-B/gastrin receptors in which one or more of residues ¹⁵¹E, ³²⁵L,and ³³²V are replaced with any other amino acid residue, e.g., a serine,aspartic acid, glutamine, or glutamic acid residue.

4. Naturally-Occurring Mutant Receptors, including but not limited tonaturally-occurring constitutively active mutant receptors, that areassociated with a disease or other adverse phenotype, e.g., a phenotypethat results from a constitutively active naturally-occurring mutantreceptor. Examples include, but are not limited to, the followingpeptide hormone receptors:

a) Point mutations in the luteinizing hormone (LH) receptor gene areresponsible for some incidences of precocious puberty. Mutant receptorsof the invention can be constructed by altering the following amino acidresidues of the LH receptor: the alanine residue at position 568 toanother amino acid, e.g., to a valine (Latronico et al., J. Clin. Endo.& Meta., 80(8):2490-94, 1995); the asparagine residue at position 578 toanother amino acid, e.g., to a glycine or a tyrosine (Kosugi et al.,Human Mol. Genet., 4(2):183-88, 1995; Laue et al., Proc. Natl. Acad.Sci. USA, 92(6):1906-10, 1995); the Met residue at position 571 toanother amino acid, e.g., to an Ile, or the Thr residue at 577 toanother amino acid residue, e.g., to an Ile (Kosugi et al., supra; Laueet al. supra); the Ile residue at position 542 to another amino acid,the Asp residue at position 564 to another amino acid, the Cys residueat position 581 to another amino acid, or the Asp residue at position578 to another amino acid (Laue et al., supra); amino acid residueswithin transmembrane helices 5 or 6, e.g., in the intracellulardomain-proximal portion of transmembrane helix 6, or in intracellularloop 3 (Laue et al. supra).

Also embraced are mutations at the corresponding residues of thefollicle stimulating hormone (FSH) receptor and the thyroid stimulatinghormone (TSH) receptor (Latronico et al., supra).

b) A naturally-occurring constitutively active parathyroid (PTH)receptor results from a His to Arg substitution at conserved position223 (Schipani et al., Science, 268:98-100, 1995). A constitutivelyactive mutant G-LP1 receptor can be constructed by substitutingalternative amino acids at the corresponding residues in relatedreceptors, e.g., substituting another amino acid for the homologue Hisin the glucagon-like peptide 1 (G-LP1) receptor. A similar change in anyof the receptors related to PTH or G-LP1 by amino acid homologyincluding, but not limited to, secretin, vasoactive intestinalpolypeptide, glucagon, G-LP1, and calcitonin.

Non-peptide positive or inverse agonists identified in a screening assayemploying any of the above-listed naturally occurring mutant receptorscan be therapeutically useful against a corresponding adverse phenotype.

5. Strategies to identify synthetic mutant receptors.

Deletional analysis defines intracellular receptor domains important insecond messenger signaling: Recombinant CCK-A and CCK-B/gastrinreceptors are both coupled to phospholipase-C activation. Applicantshypothesized that the third intracellular loop of the CCK-B/gastrinreceptor would include residues that are important in second messengersignaling. To test this hypothesis, a series of deletion mutationslocated in the third intracellular loop, each lacking between six and 55amino acids, were expressed in COS-7 cells and tested for [¹²⁵I]CCK-8binding and ³H inositol phosphate formation. Deletion of a twelve aminoacid segment in the carboxy-terminal end of the third intracellular loopresulted in normal affinity for CCK-8s, but caused a 90% reduction ofmaximal inositol phosphate formation; all other receptors in this seriessignaled normally. The region containing the twelve amino acids thatproved to functionally important was then screened for constitutivelyactive point mutations, as described below.

Strategy 1: Domain swapping with cAMP generating receptors results inconstitutive receptor activity: A method for rapidly identifyingconstitutively active mutant receptors relies on exchanging functionaldomains between two receptors, the domains being, e.g., approximately5-10 amino acids in length. One of the two receptors is a form of thereceptor which is the main template of the desired mutant receptor,e.g., a wild-type receptor; the second receptor is a different peptidehormone receptor from the first. Candidate receptors are coupled todifferent signal transduction pathways, e.g., a signal via a same ordifferent second messenger pathways, yet are closely related in theiramino acid sequence. These criteria are based on the idea that stretchesof amino acids which function normally in their native context canconfer agonist-independent signaling when transplanted into a closelyrelated receptor which is linked to a different second-messengersignaling pathway.

The domain swapping strategy was used to identify constitutively activemutants of the CCK-B/gastrin receptor. A series of short segments in thethird intracellular loop were sequentially replaced with homologousamino acid sequence from the vasopressin 2 receptor, which is thereceptor most nearly identical in sequence to hCCK-B. Vasopressin 2 isalso a good candidate for swapping domains with the CCK-B/gastrinreceptor because it is, different from the latter, linked to theadenylate cyclase signaling pathway.

309         transmembrane domain VI  359LT APGPGSGSRP TQAKLLAKKR VVRMLLVIVV LFFLCWLPVY SANTWR AFD (SEQ ID NO: 8)      AHVSA [MH40] (SEQ ID NO: 9)        SA [MH128] (SEQ ID NO: 10)       S [MH156] (SEQ ID NO: 11)        E [MH162] (SEQ ID NO: 12)

When tested, a five amino acid substitution (QAKLL (SEQ ID NO: 13 toAHVS (SEQ ID NO: 14) into the homologous position of the CCK-B/gastrinreceptor resulted in constitutive activity of the CCK-B/gastrinreceptor. The QAKLL (SEQ ID NO: 13) to AHVSA (SEQ ID NO: 14)substitution caused an increased level of basal inositol phosphateformation to 290% of the wild-type CCK-B/gastrin receptor (FIG. 9,Mutant 2). In addition, mutations causing constitutive activity includereplacement of LL to SA, L to S, and L to E.

Strategy 2: Glutamic acid scanning mutagenesis identifies constitutivelyactive receptors: In addition to, or as a substitute for, deletionanalysis or domain swapping, mutant receptors can be made using aprocess Applicants have named ‘amino acid scanning mutagenesis.’ Aminoacid scanning mutagenesis involves sequentially replacing each aminoacid found in either an intracellular loop or in the half of thetransmembrane domain flanking the intracellular portion of the receptor.An experimental option is to change the charge of the amino acid, e.g.,to exchange a negative for a positive amino acid, a positive for anegative amino acid, or a positive or negative amino acid for a neutralamino acid. Another option would be to exchange each amino acid, e.g.,each neutral amino acid, with another neutral amino acid.

In the case of the CCK-B/gastrin receptor, deletion analysis wasinitially used to define a functionally important twelve amino acidsegment within the third intracellular loop which was important forsecond messenger signaling. Subsequently, each of the neutral aminoacids within the 12 this segment was replaced sequentially with anotheramino acid, preferably with glutamic acid. The scanning analysistechnique revealed that one of the glutamic acid substitutions caused a228% increase in the basal-level of inositol phosphate accumulation,relative to the wild-type value, in transiently transfected COS-7 cells(FIG. 9, Mutant 1).

In this example, applicants focused on the region limited to the carboxyend of the third intracellular (IC) loop and the portion of the sixthtransmembrane domain which flanks the third IC loop. Glutamic acidresidues (E) were introduced in place of neutral amino acid residues.

309                    359LT APGPGSGSRP TQAKLLAKKR VVRMLLVIVV LFFLCWLPVY SANTWR AFD              |  ||   || (SEQ ID NO:15)       E E E  EE (SEQ ID NO:16)

Constitutively active receptors include an amino acid replacement of³²³A→E (MH31, SEQ ID NO: 17), ³²⁴K→E (MH131, SEQ ID NO: 18), ³²⁵L→E(MH162, SEQ ID NO: 19), ³²⁷A→E (MH13, SEQ ID NO: 20) ³³¹V→E (MH130, SEQID No: 21), ³³²V→E (129, SEQ ID NO: 22), and ³³¹VV→EE (MH72),respectively all in pcDNAI vectors, as described above.

FIG. 9 is a schematic representation of the seven transmembrane (TM)domain structure of the human CCK-B/gastrin receptor. The C-terminaldomain of the third intracellular loop, which is crucial forintracellular signaling, is highlighted in black. Within this segment,two mutations were found to confer constitutive activity on thereceptor. One of the mutations was constructed by glutamic acidsubstitution scanning (Mutant 1; MH129, SEQ ID NO: 22); a secondmutation was constructed by domain swapping (Mutant 2; MH162, SEQ ID NO:19). A bar graph showing the basal inositol phosphate accumulation inCOS-7 cells, which had been transfected with the wild-type CCK-B/gastrinreceptor or with two different constitutively active mutants, is shownin FIG. 10.

Strategy 3: A third method for making a mutant receptor is to align thereceptor of interest with a known constitutively active mutant receptor,including, but not limited to, peptide hormone, biogenic amine,rhodopsin, or other G-protein coupled receptors. An example of such analignment is shown in FIG. 2. Generally, mutations which result inconstitutive activity in the known mutant can be introduced into thecorresponding position of the receptor of interest. Examples of knownconstitutively active mutant receptors include, but are not limited to,the follicle stimulating hormone (FSH) receptor, the thyroid stimulatinghormone (TSH) receptor, and the luteinizing hormone receptor, e.g., a568 Ala to Val mutation in the LH receptor (Latronico et al., J. Clin.Endo. & Meta., 80(8):2490-94, 1995).

This method, based on alignment, was employed to construct a CCK-Amutant receptor. A multiple alignment map was made which included thehuman and rat CCK-A sequences, the mastomys, rat, human, and canineCCK-B/gastrin receptor, and a Xenopus CCK-A/CCK-B intermediate receptor(CCK-XL; FIG. 2). Based on this map, conserved amino acids 138-ERY-140of the CCK-A receptor were replaced with amino acids QRY, based on aknown constitutively active rhodopsin mutant with enhanced transducinactivation (Arnis et al., J. Biol. Chem., 269:23879-81, 1994). Thealtered amino acid residues are positioned in transmembrane domain IIIand flank the second intracellular loop. Although the basal level ofsignaling was not increased, the intrinsic activity of the non-peptideligand PD 135,158 was significantly increased.

Strategy 4: Additional mutant receptors can be made by sequentiallydeleting intracellular portions of the receptor, and looking for anincrease in basal activity, or for overactivity of a partial agonist,relative to the wild-type receptor.

Wild-type Receptors with Enhanced Basal Activity:

Peptide hormone receptors useful in the method of the invention caninclude non-human receptors which have the ability to amplify theintrinsic activity of non-peptide agonist than does the correspondinghuman wild-type receptor, or which have a higher basal level of activitythan does the human wild-type receptor.

In FIG. 11, basal levels of inositol phosphate production were measuredfor human CCK-A hCCK-A), human CCK-B (hCCK-B), dog CCK-B (dCCK-B), mouseCCK-B (mCCK-B), and the mastomys CCK receptor (FIG. 11, part A), andexpressed relative to the basal level of hCCK-B.

Single experiments were also performed for the rat CCK-B/gastrinreceptor and for the related Xenopus CCK receptor (Table 2). The human³²⁵L to E mutant served as a positive control (n=−14).

TABLE 2 CCK-8s stimulated receptor basal (% of human basal) (% humanbasal) rat CCK-A 77 684 Xenopus CCK 74 442 ³²⁵L to E CCKA 231 ± 7 771 ±36

The wild-type human CCK-A and CCK-B/gastrin receptors induced onlyinsignificant changes of basal inositol phosphate production in COS-7cells (as compared to control cells transfected with the empty plasmidvector, pCDNAI). Similarly, the wild-type rat CCK-A and canineCCK-B/gastrin receptors, as well as the closely related Xenopus CCKreceptor all appeared more or less functionally silent in the basalstate. In contrast, the wild type mouse CCK-B/gastrin receptor and itshomologue from mastomys natalensis significantly increased basalinositol phosphate production in COS-7 cells over pcDNAI controls. Whencompared with the slight basal activity of the wild type humanCCK-B/gastrin receptor, it was estimated that the basal activities ofthe wild type mouse and mastomys homologues were 7- and 11-fold higher,respectively. For comparison, the ³²⁵L→E mutant of the humanCCK-B/gastrin receptor appeared to be at least 16-fold more active thanthe human wild type receptor in its basal state. It should be noted thatthe described species differences in basal activities were clearly notrelated to different degrees of receptor expression, since the maximalresponse to stimulation with CCK-8s was comparable for all testedreceptors (positive control).

IV. Therapeutic Use.

The ability to pharmacologically modulate wild-type or constitutivelyactive receptor activity opens the door for a new class of clinicallyuseful drugs. Enhanced receptors will enable the discovery of noveldrugs directed at a broad spectrum of diseases. Constitutively activemutants of the thyrotropin, luteinizing hormone, and parathyroid hormonereceptors are already known to occur in nature (see above) and mightprovide a starting point for non-peptide agonist/inverse agonistscreening. For example, drugs which silence constitutively activethyroid stimulating hormone receptors, which are implicated in theetiology of thyroid adenomas, could be used to inhibit tumor growth.Similarly, in patients with constitutively active luteinizing hormonereceptors, inverse agonists could delay the onset of precocious puberty.

Further information on peptide hormone receptor amino acid sequences,receptor-specific agonists and antagonists, receptor conformation,pharmacology, receptor-encoding genes, animal models for subsequentfollow-up studies, and database accession numbers can be obtained from:Watson and Arkinstall, The G-Protein Linked Receptor, Academic Press,NY., 1994; see also, Kolakowski, L. F., “The G Protein-Coupled ReceptorDatabase”, World-Wide-Web Site, GCRDB-WWW.

OTHER EMBODIMENTS

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

Other embodiments are within the following claims.

23 449 amino acids amino acid Not Relevant linear protein 1 Met Glu LeuLeu Lys Leu Asn Ser Ser Val Gln Gly Pro Gly Pro Gly 1 5 10 15 Ser GlySer Ser Leu Cys His Pro Gly Val Ser Leu Leu Asn Ser Ser 20 25 30 Ala GlyAsn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Thr Gly Thr 35 40 45 Arg GluLeu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile Phe 50 55 60 Leu MetSer Ile Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly Leu 65 70 75 80 SerArg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu Ala 85 90 95 ValSer Asp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu Leu 100 105 110Pro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys Ala 115 120125 Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Asn Leu 130135 140 Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu Gln145 150 155 160 Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val IleLeu Ala 165 170 175 Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr ProVal Tyr Thr 180 185 190 Val Val Gln Pro Val Gly Pro Arg Val Leu Gln CysMet His Arg Trp 195 200 205 Pro Ser Ala Arg Val Arg Gln Thr Trp Ser ValLeu Leu Leu Met Leu 210 215 220 Leu Phe Phe Ile Pro Gly Val Val Met AlaVal Ala Tyr Gly Leu Ile 225 230 235 240 Ser Arg Glu Leu Tyr Leu Gly LeuArg Phe Asp Gly Asp Asn Asp Ser 245 250 255 Asp Thr Gln Ser Arg Val ArgAsn Gln Gly Gly Leu Pro Gly Gly Thr 260 265 270 Ala Pro Gly Pro Val HisGln Asn Gly Gly Cys Arg His Val Thr Val 275 280 285 Ala Gly Glu Asp AsnAsp Gly Cys Tyr Val Gln Leu Pro Arg Ser Arg 290 295 300 Leu Glu Met ThrThr Leu Thr Thr Pro Thr Pro Gly Pro Gly Leu Ala 305 310 315 320 Ser AlaAsn Gln Ala Lys Leu Leu Ala Lys Lys Arg Val Val Arg Met 325 330 335 LeuLeu Val Ile Val Leu Leu Phe Phe Leu Cys Trp Leu Pro Ile Tyr 340 345 350Ser Ala Asn Thr Trp Cys Ala Phe Asp Gly Pro Gly Ala Met Arg Ala 355 360365 Leu Ser Gly Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser 370375 380 Ala Cys Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg385 390 395 400 Gln Ala Cys Leu Asp Thr Cys Ala Arg Cys Cys Pro Arg ProPro Arg 405 410 415 Ala Arg Pro Arg Pro Leu Pro Asp Glu Asp Pro Pro ThrPro Ser Ile 420 425 430 Ala Ser Leu Ser Arg Leu Ser Tyr Thr Thr Ile SerThr Leu Gly Pro 435 440 445 Gly 451 amino acids amino acid Not Relevantlinear protein 2 Met Glu Leu Leu Lys Leu Asn Arg Ser Val Gln Gly Pro GlyPro Gly 1 5 10 15 Ser Gly Ser Ser Leu Cys Arg Pro Gly Val Ser Leu LeuAsn Ser Ser 20 25 30 Ser Ala Gly Asn Leu Ser Cys Asp Pro Pro Arg Ile ArgGly Thr Gly 35 40 45 Thr Arg Glu Leu Glu Met Ala Ile Arg Ile Thr Leu TyrAla Val Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Val Leu Ile Ile ValVal Leu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala PheLeu Leu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys MetPro Phe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile Phe GlyThr Val Ile Cys Lys 115 120 125 Ala Ile Ser Tyr Leu Met Gly Val Ser ValSer Val Ser Thr Leu Asn 130 135 140 Leu Val Ala Ile Ala Leu Glu Arg TyrSer Ala Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala Arg Val Trp Gln ThrArg Ser His Ala Ala Arg Val Ile Leu 165 170 175 Ala Thr Trp Leu Leu SerGly Leu Leu Met Val Pro Tyr Pro Val Tyr 180 185 190 Thr Met Val Gln ProVal Gly Pro Arg Val Leu Gln Cys Met His Arg 195 200 205 Trp Pro Ser AlaArg Val Gln Gln Thr Trp Ser Val Leu Leu Leu Leu 210 215 220 Leu Leu PhePhe Ile Pro Gly Val Val Ile Ala Val Ala Tyr Gly Leu 225 230 235 240 IleSer Arg Glu Leu Tyr Leu Gly Leu His Phe Asp Gly Glu Asn Asp 245 250 255Ser Glu Thr Gln Ser Arg Ala Arg Asn Gln Gly Gly Leu Pro Gly Gly 260 265270 Ala Ala Pro Gly Pro Val His Gln Asn Gly Gly Cys Arg Pro Val Thr 275280 285 Ser Val Ala Gly Glu Asp Ser Asp Gly Cys Cys Val Gln Leu Pro Arg290 295 300 Ser Arg Leu Glu Met Thr Thr Leu Thr Thr Pro Thr Gly Pro ValPro 305 310 315 320 Gly Pro Arg Pro Asn Gln Ala Lys Leu Leu Ala Lys LysArg Val Val 325 330 335 Arg Met Leu Leu Val Ile Val Leu Leu Phe Phe LeuCys Trp Leu Pro 340 345 350 Val Tyr Ser Val Asn Thr Trp Arg Ala Phe AspGly Pro Gly Ala Gln 355 360 365 Arg Ala Leu Ser Gly Ala Pro Ile Ser PheIle His Leu Leu Ser Tyr 370 375 380 Val Ser Ala Cys Val Asn Pro Leu ValTyr Cys Phe Met His Arg Arg 385 390 395 400 Phe Arg Gln Ala Cys Leu AspThr Cys Ala Arg Cys Cys Pro Arg Pro 405 410 415 Pro Arg Ala Arg Pro GlnPro Leu Pro Asp Glu Asp Pro Pro Thr Pro 420 425 430 Ser Ile Ala Ser LeuSer Arg Leu Ser Tyr Thr Thr Ile Ser Thr Leu 435 440 445 Gly Pro Gly 450448 amino acids amino acid Not Relevant linear protein 3 Met Glu Leu LeuLys Leu Asn Arg Ser Val Gln Gly Thr Gly Pro Gly 1 5 10 15 Pro Gly AlaSer Leu Cys Arg Pro Gly Ala Pro Leu Leu Asn Ser Ser 20 25 30 Ser Val GlyAsn Leu Ser Cys Glu Pro Pro Arg Ile Arg Gly Ala Gly 35 40 45 Thr Arg GluLeu Glu Leu Ala Ile Arg Ile Thr Leu Tyr Ala Val Ile 50 55 60 Phe Leu MetSer Val Gly Gly Asn Met Leu Ile Ile Val Val Leu Gly 65 70 75 80 Leu SerArg Arg Leu Arg Thr Val Thr Asn Ala Phe Leu Leu Ser Leu 85 90 95 Ala ValSer Asp Leu Leu Leu Ala Val Ala Cys Met Pro Phe Thr Leu 100 105 110 LeuPro Asn Leu Met Gly Thr Phe Ile Phe Gly Thr Val Ile Cys Lys 115 120 125Ala Val Ser Tyr Leu Met Gly Val Ser Val Ser Val Ser Thr Leu Ser 130 135140 Leu Val Ala Ile Ala Leu Glu Arg Tyr Ser Ala Ile Cys Arg Pro Leu 145150 155 160 Gln Ala Arg Val Trp Gln Thr Arg Ser His Ala Ala Arg Val IleVal 165 170 175 Ala Thr Trp Leu Leu Ser Gly Leu Leu Met Val Pro Tyr ProVal Tyr 180 185 190 Thr Val Val Gln Pro Val Gly Pro Arg Val Leu Gln CysVal His Arg 195 200 205 Trp Pro Ser Ala Arg Val Arg Gln Thr Trp Ser ValLeu Leu Leu Leu 210 215 220 Leu Leu Phe Phe Ile Pro Gly Val Val Met AlaVal Ala Tyr Gly Leu 225 230 235 240 Ile Ser Arg Glu Leu Tyr Leu Gly LeuArg Phe Asp Gly Asp Ser Asp 245 250 255 Ser Asp Ser Gln Ser Arg Val ArgAsn Gln Gly Gly Leu Pro Gly Ala 260 265 270 Val His Gln Asn Gly Arg CysArg Pro Glu Thr Gly Ala Val Gly Glu 275 280 285 Asp Ser Asp Gly Cys TyrVal Gln Leu Pro Arg Ser Arg Pro Ala Leu 290 295 300 Glu Leu Thr Ala LeuThr Ala Pro Gly Pro Gly Gly Ser Gly Ser Arg 305 310 315 320 Pro Thr GlnAla Lys Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu 325 330 335 Leu ValIle Val Val Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser 340 345 350 AlaAsn Thr Trp Arg Ala Phe Asp Gly Pro Gly Ala His Arg Ala Leu 355 360 365Ser Gly Ala Pro Ile Ser Phe Ile His Leu Leu Ser Tyr Ala Ser Ala 370 375380 Cys Val Asn Pro Leu Val Tyr Cys Phe Met His Arg Arg Phe Arg Gln 385390 395 400 Ala Cys Leu Glu Thr Cys Ala Arg Cys Cys Pro Arg Pro Pro ArgAla 405 410 415 Arg Pro Arg Ala Leu Pro Asp Glu Asp Pro Pro Thr Pro SerIle Ala 420 425 430 Ser Leu Ser Arg Leu Ser Tyr Thr Thr Ile Ser Thr LeuGly Pro Gly 435 440 445 453 amino acids amino acid Not Relevant linearprotein 4 Met Glu Leu Leu Lys Leu Asn Arg Ser Ala Gln Gly Ser Gly AlaGly 1 5 10 15 Pro Gly Ala Ser Leu Cys Arg Ala Gly Gly Ala Leu Leu AsnSer Ser 20 25 30 Gly Ala Gly Asn Leu Ser Cys Glu Pro Pro Arg Leu Arg GlyAla Gly 35 40 45 Thr Arg Glu Leu Glu Leu Ala Ile Arg Val Thr Leu Tyr AlaVal Ile 50 55 60 Phe Leu Met Ser Val Gly Gly Asn Val Leu Ile Ile Val ValLeu Gly 65 70 75 80 Leu Ser Arg Arg Leu Arg Thr Val Thr Asn Ala Phe LeuLeu Ser Leu 85 90 95 Ala Val Ser Asp Leu Leu Leu Ala Val Ala Cys Met ProPhe Thr Leu 100 105 110 Leu Pro Asn Leu Met Gly Thr Phe Ile Phe Gly ThrVal Val Cys Lys 115 120 125 Ala Val Ser Tyr Leu Met Gly Val Ser Val SerVal Ser Thr Leu Ser 130 135 140 Leu Val Ala Ile Ala Leu Glu Arg Tyr SerAla Ile Cys Arg Pro Leu 145 150 155 160 Gln Ala Arg Val Trp Gln Thr ArgSer His Ala Ala Arg Val Ile Ile 165 170 175 Ala Thr Trp Met Leu Ser GlyLeu Leu Met Val Pro Tyr Pro Val Tyr 180 185 190 Thr Ala Val Gln Pro AlaGly Gly Ala Arg Ala Leu Gln Cys Val His 195 200 205 Arg Trp Pro Ser AlaArg Val Arg Gln Thr Trp Ser Val Leu Leu Leu 210 215 220 Leu Leu Leu PhePhe Val Pro Gly Val Val Met Ala Val Ala Tyr Gly 225 230 235 240 Leu IleSer Arg Glu Leu Tyr Leu Gly Leu Arg Phe Asp Glu Asp Ser 245 250 255 AspSer Glu Ser Arg Val Arg Ser Gln Gly Gly Leu Arg Gly Gly Ala 260 265 270Gly Pro Gly Pro Ala Pro Pro Asn Gly Ser Cys Arg Pro Glu Gly Gly 275 280285 Leu Ala Gly Glu Asp Gly Asp Gly Cys Tyr Val Gln Leu Pro Arg Ser 290295 300 Arg Gln Thr Leu Glu Leu Ser Ala Leu Thr Ala Pro Thr Pro Gly Pro305 310 315 320 Gly Gly Gly Pro Arg Pro Tyr Gln Ala Lys Leu Leu Ala LysLys Arg 325 330 335 Val Val Arg Met Leu Leu Val Ile Val Val Leu Phe PheLeu Cys Trp 340 345 350 Leu Pro Leu Tyr Ser Ala Asn Thr Trp Arg Ala PheAsp Ser Ser Gly 355 360 365 Ala His Arg Ala Leu Ser Gly Ala Pro Ile SerPhe Ile His Leu Leu 370 375 380 Ser Tyr Ala Ser Ala Cys Val Asn Pro LeuVal Tyr Cys Phe Met His 385 390 395 400 Arg Arg Phe Arg Gln Ala Cys LeuGlu Thr Cys Ala Arg Cys Cys Pro 405 410 415 Arg Pro Pro Arg Ala Arg ProArg Pro Leu Pro Asp Glu Asp Pro Pro 420 425 430 Thr Pro Ser Ile Ala SerLeu Ser Arg Leu Ser Tyr Thr Thr Ile Ser 435 440 445 Thr Leu Gly Pro Gly450 428 amino acids amino acid Not Relevant linear protein 5 Met Asp ValVal Asp Ser Leu Leu Val Asn Gly Ser Asn Ile Thr Pro 1 5 10 15 Pro CysGlu Leu Gly Leu Glu Asn Glu Thr Leu Phe Cys Leu Asp Gln 20 25 30 Pro ArgPro Ser Lys Glu Trp Gln Pro Ala Val Gln Ile Leu Leu Tyr 35 40 45 Ser LeuIle Phe Leu Leu Ser Val Leu Gly Asn Thr Leu Val Ile Thr 50 55 60 Val LeuIle Arg Asn Lys Arg Met Arg Thr Val Thr Asn Ile Phe Leu 65 70 75 80 LeuSer Leu Ala Val Ser Asp Leu Met Leu Cys Leu Phe Cys Met Pro 85 90 95 PheAsn Leu Ile Pro Asn Leu Leu Lys Asp Phe Ile Phe Gly Ser Ala 100 105 110Val Cys Lys Thr Thr Thr Tyr Phe Met Gly Thr Ser Val Ser Val Ser 115 120125 Thr Phe Asn Leu Val Ala Ile Ser Leu Glu Arg Tyr Gly Ala Ile Cys 130135 140 Lys Pro Leu Gln Ser Arg Val Trp Gln Thr Lys Ser His Ala Leu Lys145 150 155 160 Val Ile Ala Ala Thr Trp Cys Leu Ser Phe Thr Ile Met ThrPro Tyr 165 170 175 Pro Ile Tyr Ser Asn Leu Val Pro Phe Thr Lys Asn AsnAsn Gln Thr 180 185 190 Ala Asn Met Cys Arg Phe Leu Leu Pro Asn Asp ValMet Gln Gln Ser 195 200 205 Trp His Thr Phe Leu Leu Leu Ile Leu Phe LeuIle Pro Gly Ile Val 210 215 220 Met Met Val Ala Tyr Gly Leu Ile Ser LeuGlu Leu Tyr Gln Gly Ile 225 230 235 240 Lys Phe Glu Ala Ser Gln Lys LysSer Ala Lys Glu Arg Lys Pro Ser 245 250 255 Thr Thr Ser Ser Gly Lys TyrGlu Asp Ser Asp Gly Cys Tyr Leu Gln 260 265 270 Lys Thr Arg Pro Pro ArgLys Leu Glu Leu Arg Gln Leu Ser Thr Gly 275 280 285 Ser Ser Ser Arg AlaAsn Arg Ile Arg Ser Asn Ser Ser Ala Ala Asn 290 295 300 Leu Met Ala LysLys Arg Val Ile Arg Met Leu Ile Val Ile Val Val 305 310 315 320 Leu PhePhe Leu Cys Trp Met Pro Ile Phe Ser Ala Asn Ala Trp Arg 325 330 335 AlaTyr Asp Thr Ala Ser Ala Glu Arg Arg Leu Ser Gly Thr Pro Ile 340 345 350Ser Phe Ile Leu Leu Leu Ser Tyr Thr Ser Ser Cys Val Asn Pro Ile 355 360365 Ile Tyr Cys Phe Met Asn Lys Arg Phe Arg Leu Gly Phe Met Ala Thr 370375 380 Phe Pro Cys Cys Pro Asn Pro Gly Pro Pro Gly Ala Arg Gly Glu Val385 390 395 400 Gly Glu Glu Glu Glu Gly Gly Thr Thr Gly Ala Ser Leu SerArg Phe 405 410 415 Ser Tyr Ser His Met Ser Ala Ser Val Pro Pro Gln 420425 443 amino acids amino acid Not Relevant linear protein 6 Met Ser HisSer Pro Ala Arg Gln His Leu Val Glu Ser Ser Arg Met 1 5 10 15 Asp ValVal Asp Ser Leu Leu Met Asn Gly Ser Asn Ile Thr Pro Pro 20 25 30 Cys GluLeu Gly Leu Glu Asn Glu Thr Leu Phe Cys Leu Asp Gln Pro 35 40 45 Gln ProSer Lys Glu Trp Gln Ser Ala Leu Gln Ile Leu Leu Tyr Ser 50 55 60 Ile IlePhe Leu Leu Ser Val Leu Gly Asn Thr Leu Val Ile Thr Val 65 70 75 80 LeuIle Arg Asn Lys Arg Met Arg Thr Val Thr Asn Ile Phe Leu Leu 85 90 95 SerLeu Ala Val Ser Asp Leu Met Leu Cys Phe Cys Met Pro Phe Asn 100 105 110Leu Ile Pro Asn Leu Leu Lys Asp Phe Ile Phe Gly Ser Ala Val Cys 115 120125 Lys Thr Thr Thr Tyr Phe Met Gly Thr Ser Val Ser Val Ser Thr Phe 130135 140 Asn Leu Val Ala Ile Ser Leu Glu Arg Tyr Gly Ala Ile Cys Arg Pro145 150 155 160 Leu Gln Ser Arg Val Trp Gln Thr Lys Ser His Ala Leu LysVal Ile 165 170 175 Ala Ala Thr Trp Cys Leu Ser Phe Thr Ile Met Thr ProTyr Pro Ile 180 185 190 Tyr Ser Asn Leu Val Pro Phe Thr Lys Asn Asn AsnGln Thr Ala Asn 195 200 205 Met Cys Arg Phe Leu Leu Pro Ser Asp Ala MetGln Gln Ser Trp Gln 210 215 220 Thr Phe Leu Leu Leu Ile Leu Phe Leu LeuPro Gly Ile Val Met Val 225 230 235 240 Val Ala Tyr Gly Leu Ile Ser LeuGlu Leu Tyr Gln Gly Ile Lys Phe 245 250 255 Asp Ala Ser Gln Lys Lys SerAla Lys Glu Lys Lys Pro Ser Thr Gly 260 265 270 Ser Ser Thr Arg Tyr GluAsp Ser Asp Gly Cys Tyr Leu Gln Lys Ser 275 280 285 Arg Pro Pro Arg LysLeu Glu Leu Gln Gln Leu Ser Ser Gly Ser Gly 290 295 300 Gly Ser Arg LeuAsn Arg Ile Arg Ser Ser Ser Ser Ala Ala Asn Leu 305 310 315 320 Ile AlaLys Lys Arg Val Ile Arg Met Leu Ile Val Ile Val Val Leu 325 330 335 PhePhe Leu Cys Trp Met Pro Ile Phe Ser Ala Asn Ala Trp Arg Ala 340 345 350Tyr Asp Thr Val Ser Ala Glu Lys His Leu Ser Gly Thr Pro Ile Ser 355 360365 Phe Ile Leu Leu Leu Ser Tyr Thr Ser Ser Cys Val Asn Pro Ile Ile 370375 380 Tyr Cys Phe Met Asn Lys Arg Phe Arg Leu Gly Phe Met Ala Thr Phe385 390 395 400 Pro Cys Cys Pro Asn Pro Gly Pro Pro Gly Val Arg Gly GluVal Gly 405 410 415 Glu Glu Glu Asp Gly Arg Thr Ile Arg Ala Leu Leu SerArg Tyr Ser 420 425 430 Tyr Ser His Met Ser Thr Ser Ala Pro Pro Pro 435440 453 amino acids amino acid Not Relevant linear protein 7 Met Glu SerLeu Arg Ser Leu Ser Asn Ile Ser Ala Leu His Glu Leu 1 5 10 15 Leu CysArg Tyr Ser Asn Leu Ser Gly Thr Leu Thr Trp Asn Leu Ser 20 25 30 Ser ThrAsn Gly Thr His Asn Leu Thr Thr Ala Asn Trp Pro Pro Trp 35 40 45 Asn LeuAsn Cys Thr Pro Ile Leu Asp Arg Lys Lys Pro Ser Pro Ser 50 55 60 Asp LeuAsn Leu Trp Val Arg Ile Val Met Tyr Ser Val Ile Phe Leu 65 70 75 80 LeuSer Val Phe Gly Asn Thr Leu Ile Ile Ile Val Leu Val Met Asn 85 90 95 LysArg Leu Arg Thr Ile Thr Asn Ser Phe Leu Leu Ser Leu Ala Leu 100 105 110Ser Asp Leu Met Val Ala Val Leu Cys Met Pro Phe Thr Leu Ile Pro 115 120125 Asn Leu Met Glu Asn Phe Ile Phe Gly Glu Val Ile Cys Arg Ala Ala 130135 140 Ala Tyr Phe Met Gly Leu Ser Val Ser Val Ser Thr Phe Asn Leu Val145 150 155 160 Ala Ile Ser Ile Glu Arg Tyr Ser Ala Ile Cys Asn Pro LeuXaa Ser 165 170 175 Arg Val Trp Gln Thr Arg Ser His Ala Tyr Arg Val IleAla Ala Thr 180 185 190 Trp Val Leu Ser Ser Ile Ile Met Ile Pro Tyr LeuVal Tyr Asn Lys 195 200 205 Thr Val Thr Phe Pro Met Lys Asp Arg Arg ValGly His Gln Cys Arg 210 215 220 Leu Val Trp Pro Ser Lys Gln Val Gln GlnAla Trp Tyr Val Leu Leu 225 230 235 240 Leu Thr Ile Leu Phe Phe Ile ProGly Val Val Met Ile Val Ala Tyr 245 250 255 Gly Leu Ile Ser Arg Glu LeuTyr Arg Gly Ile Gln Phe Glu Met Asp 260 265 270 Leu Asn Lys Glu Ala LysAla His Lys Asn Gly Val Ser Thr Pro Thr 275 280 285 Thr Ile Pro Ser GlyAsp Glu Gly Asp Gly Cys Tyr Ile Gln Val Thr 290 295 300 Lys Arg Arg AsnThr Met Glu Met Ser Thr Leu Thr Pro Ser Val Cys 305 310 315 320 Thr LysMet Asp Arg Ala Arg Ile Asn Asn Ser Glu Ala Lys Leu Met 325 330 335 AlaLys Lys Arg Val Ile Arg Met Leu Ile Val Ile Val Ala Met Phe 340 345 350Phe Ile Cys Trp Met Pro Ile Phe Val Ala Asn Thr Trp Lys Ala Phe 355 360365 Asp Glu Leu Ser Ala Phe Asn Thr Leu Thr Gly Ala Pro Ile Ser Phe 370375 380 Ile His Leu Leu Ser Tyr Thr Ser Ala Cys Val Asn Pro Leu Ile Tyr385 390 395 400 Cys Phe Met Asn Lys Arg Phe Arg Lys Ala Phe Leu Gly ThrPhe Ser 405 410 415 Ser Cys Ile Lys Pro Cys Arg Asn Phe Arg Asp Thr AspGlu Asp Ile 420 425 430 Ala Ala Thr Gly Ala Ser Leu Ser Lys Phe Ser TyrThr Thr Val Ser 435 440 445 Ser Leu Gly Pro Ala 450 51 amino acids aminoacid Not Relevant linear protein 8 Leu Thr Ala Pro Gly Pro Gly Ser GlySer Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Ala Lys Lys Arg Val ValArg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu Cys Trp Leu ProVal Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51 amino acidsamino acid Not Relevant linear protein 9 Leu Thr Ala Pro Gly Pro Gly SerGly Ser Arg Pro Thr Ala His Val 1 5 10 15 Ser Ala Ala Lys Lys Arg ValVal Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu Cys Trp LeuPro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51 aminoacids amino acid Not Relevant linear protein 10 Leu Thr Ala Pro Gly ProGly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Ser Ala Ala Lys LysArg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu CysTrp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51amino acids amino acid Not Relevant linear protein 11 Leu Thr Ala ProGly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Ser Leu AlaLys Lys Arg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe PheLeu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp50 51 amino acids amino acid Not Relevant linear protein 12 Leu Thr AlaPro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Glu LeuAla Lys Lys Arg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu PhePhe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala PheAsp 50 5 amino acids amino acid Not Relevant linear protein 13 Gln AlaLys Leu Leu 1 5 5 amino acids amino acid Not Relevant linear protein 14Ala His Tyr Ser Ala 1 5 51 amino acids amino acid Not Relevant linearprotein 15 Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln AlaLys 1 5 10 15 Leu Leu Ala Lys Lys Arg Val Val Arg Met Leu Leu Val IleVal Val 20 25 30 Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn ThrTrp Arg 35 40 45 Ala Phe Asp 50 51 amino acids amino acid Not Relevantlinear protein 16 Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser Arg Pro ThrGln Glu Lys 1 5 10 15 Glu Leu Glu Lys Lys Arg Glu Glu Arg Met Leu LeuVal Ile Val Val 20 25 30 Leu Phe Phe Leu Cys Trp Leu Pro Val Tyr Ser AlaAsn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51 amino acids amino acid NotRelevant linear protein 17 Leu Thr Ala Pro Gly Pro Gly Ser Gly Ser ArgPro Thr Gln Glu Lys 1 5 10 15 Leu Leu Ala Lys Lys Arg Val Val Arg MetLeu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu Cys Trp Leu Pro Val TyrSer Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51 amino acids aminoacid Not Relevant linear protein 18 Leu Thr Ala Pro Gly Pro Gly Ser GlySer Arg Pro Thr Gln Ala Glu 1 5 10 15 Leu Leu Ala Lys Lys Arg Val ValArg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu Cys Trp Leu ProVal Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51 amino acidsamino acid Not Relevant linear protein 19 Leu Thr Ala Pro Gly Pro GlySer Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Glu Leu Ala Lys Lys ArgVal Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu Cys TrpLeu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51 aminoacids amino acid Not Relevant linear protein 20 Leu Thr Ala Pro Gly ProGly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu Glu Lys LysArg Val Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe Phe Leu CysTrp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp 50 51amino acids amino acid Not Relevant linear protein 21 Leu Thr Ala ProGly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu Leu AlaLys Lys Arg Glu Val Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu Phe PheLeu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala Phe Asp50 51 amino acids amino acid Not Relevant linear protein 22 Leu Thr AlaPro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 Leu LeuAla Lys Lys Arg Val Glu Arg Met Leu Leu Val Ile Val Val 20 25 30 Leu PhePhe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 Ala PheAsp 50 51 amino acids amino acid Not Relevant linear protein 23 Leu ThrAla Pro Gly Pro Gly Ser Gly Ser Arg Pro Thr Gln Ala Lys 1 5 10 15 LeuLeu Ala Lys Lys Arg Glu Glu Arg Met Leu Leu Val Ile Val Val 20 25 30 LeuPhe Phe Leu Cys Trp Leu Pro Val Tyr Ser Ala Asn Thr Trp Arg 35 40 45 AlaPhe Asp 50

What is claimed is:
 1. A screening method comprising: (a) providing anenhanced chimeric G protein-coupled peptide hormone receptor thatsignals through the same pathway as a respective wild-type Gprotein-coupled receptor, in which a region of a functional domain of afirst peptide hormone receptor is replaced with a corresponding regionof a functional domain of a second peptide hormone receptor, whereinsaid functional domain is selected from the group consisting of anintracellular loop and a transmembrane domain; wherein the ability ofsaid chimeric peptide hormone receptor to amplify an agonist signal isgreater than the ability of the first peptide hormone receptor toamplify said agonist signal; and (b) using said chimeric peptide hormonereceptor to screen ligands for agonist activity.
 2. The method of claim1, wherein the first peptide hormone receptor is a CCK-B/gastrinreceptor.
 3. The method of claim 1, wherein the second peptide hormonereceptor is a vasopressin 2 receptor.
 4. The method of claim 1, whereinthe chimeric receptor has, in place of a functional domain of aCCK-B/gastrin receptor, a functional domain of a vasopressin 2 receptor.5. The method of claim 4, wherein said functional domain of saidCCK-B/gastrin receptor comprises the third intracellular loop and theamino acid sequence QAKLL (SEQ ID NO: 13) found in said thirdintracellular loop is replaced with the amino acid sequence AHVSA (SEQID NO: 9).
 6. The method of claim 4, wherein said functional domain ofsaid CCK-B/gastrin receptor comprises the amino acid sequence found atpositions 322 to 326 of the human CCK-B/gastrin receptor (SEQ ID NO: 3)and said amino acid sequence is replaced with the amino acid sequenceAHVSA (SEQ ID NO: 9).
 7. The method of claim 4, wherein said functionaldomain of said CCK-B/gastrin receptor comprises the amino acid found atposition 325 to 326 of the human CCK-B/gastrin receptor (SEQ ID NO: 3)and said amino acid is replaced with an amino acid sequence SA.
 8. Themethod of claim 4, wherein said functional domain of said CCK-B/gastrinreceptor comprises the amino acid found at position 325 of the humanCCK-B/gastrin receptor (SEQ ID NO: 3) and said amino acid is replacedwith an amino acid selected from the group consisting of S and E.